Genetic and pharmacological regulation of DYRK2 in chronic myeloid leukemia
Introduction
Chronic myeloid leukemia (CML) is a slow-progression hematological malignancy driven by the oncoprotein BCR-ABL1, with activated tyrosine kinase, resulting from the chromosomal translocation t(9;22). CML is successfully managed by inhibiting BCR-ABL1 with tyrosine kinase inhibitors (TKIs); however, most patients must remain on lifelong therapy because this treatment cannot efficiently eradicate leukemic stem cells (LSCs), a rare leukemic population responsible for chemoresistance and relapses. The induction of treatment-free remissions will require the development of LSC-specific drugs. We reported that induced expression of the dual-specificity kinase DYRK2 either by genetic loss of the Krüppel-like factor 4 (KLF4) or inhibition of the ubiquitin ligase SIAH2 with menadione (vitamin K3) abrogates the self-renewal and survival of CML LSCs. The pleiotropic effects and toxicity to red blood cells of menadione preclude its clinical translation, highlighting the need for novel agents to stabilize/activate DYRK2. Based on these findings, we propose that DYRK2 inhibits survival and self-renewal of LSCs through p53 activation and c-Myc depletion, and thus pharmacological upregulation of DYRK2 represents a novel therapeutic approach.
Methods
Herein, we show genetic validation of DYRK2 anti-leukemic property in the BCR-ABL1 mouse model of CML and screening drugs and pathway inhibitors to elucidate DYRK2 regulation and identify drugs with anti-CML capacity. Retroviral co-transduction hematopoietic cells with retroviruses carrying DYRK2 and BCR-ABL1 were used to determine the effect of DYRK2 upregulation on CML progression. To study pharmacological DYRK2 activation, we performed cytotoxicity assays and immunoblots using a panel of CML cell lines (e.g., K562, KCL-22, KU812).
Results
Retroviral DYRK2 expression in BCR-ABL1 leukemic cells inhibited the in vivo expansion of leukemia, further validating DYRK2 as a therapeutic target in a CML mouse model. Then, we investigated pharmacological DYRK2 upregulation with a panel of drugs targeting different cellular mechanisms: KLF4 inhibition (Kenpaullone), SIAH2 inhibition (Adapalene, Betulinic acid), DNA damage (Etoposide, Cisplatin, Vincristine), MDM2 inhibition (SP-141, AMG232), and proteasome inhibition (Bortezomib). This screen revealed dose-dependent cytotoxicity in a panel of CML cell lines with IC50s ranging from 1 to 45 μM. The cytotoxicity of these drugs was due to increased apoptosis and was associated with the upregulation of the DYRK2 protein. These findings support our model that DYRK2 expression is transcriptionally regulated by KLF4 and post-transcriptionally regulated by proteasomal degradation and DNA damage response.
Conclusion
CPRIT previously funded this project. Our findings support the model that genetic or pharmacological DYRK2 upregulation induces apoptosis in CML cells by activating p53 and depleting c-MYC. As a future direction, we plan to monitor DYRK2 expression in a dual-luciferase reporter system in a screen NCI drug panels (diversity set VII and approved oncology drugs) and high-throughput screen of chemical libraries containing approved drugs, investigational agents, and bioactive molecules. This work proposes DYRK2 as a novel therapeutic target and provides small molecules inducing DYRK2 expression and inhibiting CML cell survival.